Mis12 Complex Research Articles

Determining the Role of Oxysterol Binding Protein‐Related Protein 4 (ORP4) in Cell Proliferation and Survival

Oxysterol binding protein (OSBP) and related proteins (ORPs) constitute a 12-member family of mammalian lipid binding proteins with diverse cellular expression, localization and ligands. ORP4 is closely related to OSBP and expressed as short (ORP4S), medium (ORP4M) and long (ORP4L) isoforms as a result of alternate transcription start sites. ORP4 was initially discovered in disseminated tumor cells and considered a potential marker of metastasis. We recently showed that lentiviral short-hairpin RNA silencing of ORP4 induced cell-specific growth arrest or apoptosis. To determine how ORP4 promotes cell proliferation and survival, we identified relevant signaling pathways that are activated upon ORP4 silencing. In HeLa cells, ORP4 silencing was accompanied by elevated expression of p21 and phospho-p53 isoforms as well as cell cycle arrest but not apoptosis. ORP4 silencing also induced growth arrest in HEK 293 cells but triggered apoptosis in non-transformed rat intestinal epithelial cells. A potential role for ORP4L in genomic stability is indicated by its interaction with a component of the Mis18 complex required for centromere organization. Mass spectrometry was used to identify a MAPK phosphorylation site in the C-terminal lipid-binding domain of ORP4L. An ORP4L phospho-mimetic (serine-to-glutamate) MAPK site mutant expressed in HeLa cells was associated with an aggregated vimentin network, which was not observed with the corresponding serine-to-alanine mutant or wild-type ORP4L. These data suggest that ORP4L phosphorylation by MAPK enhances its interaction with the cytoskeleton and/or genomic regulators, ultimately affecting p21/p53-dependent control of cell cycle progression.

Distinct Organization and Regulation of the Outer Kinetochore KMN Network Downstream of CENP-C and CENP-T

SummaryThe kinetochore provides a vital connection between chromosomes and spindle microtubules [1, 2]. Defining the molecular architecture of the core kinetochore components is critical for understanding the mechanisms by which the kinetochore directs chromosome segregation. The KNL1/Mis12 complex/Ndc80 complex (KMN) network acts as the primary microtubule binding interface at kinetochores [3], and provides a platform to recruit regulatory proteins [4]. Recent work found that the inner kinetochore components CENP-C and CENP-T act in parallel to recruit the KMN network to kinetochores [5-8]. However, due to the presence of these dual pathways, it has not been possible to distinguish differences in the nature of kinetochore assembly downstream of CENP-C or CENP-T. Here, we separated these pathways by targeting CENP-C and CENP-T independently to an ectopic chromosomal locus in human cells. Our work reveals that the organization of the KMN network components downstream of CENP-C and CENP-T is distinct. CENP-C recruits the Ndc80 complex through its interactions with KNL1 and the Mis12 complex. In contrast, CENP-T directly interacts with Ndc80, which in turn promotes KNL1/Mis12 complex recruitment through a separate region on CENP-T, resulting in functional relationships for KMN network localization that are inverted relative to the CENP-C pathway. We also find that distinct regulatory paradigms control the assembly of these pathways, with Aurora B kinase promoting KMN network recruitment to CENP-C, and cyclin-dependent kinase (CDK) regulating KMN network recruitment to CENP-T. This work reveals unexpected complexity for the architecture and regulation of the core components of the kinetochore-microtubule interface.

Cdk1: conductor of CENP-A symphony orchestra

The inheritance of human genome through mitosis is precisely regulated by a series of tightly orchestrated events such as chromosome condensation, bi-orientated spindle formation, chromosome congression, segregation and cytokinesis. Chromosome movements during mitosis are governed by dynamic interactions of spindle microtubules with a specialized chromosome domain called centromere. Centromere provides the platform for the assembly of a protein machine named kinetochore that orchestrates accurate interaction between chromosomes and spindle microtubules during cell division. In eukaryotic organisms, the centromere is epigenetically specified by nucleosomes containing a unique histone H3 variant CENP-A, with the exception of budding yeast [1]. CENP-A orthologues present in all known eukaryotes. It is the most important epigenetic marker of centromere [2] and is required for the correct localization of most centromereand kinetochorerelated proteins [2]. CENP-A is the epigenetic marker to maintain and propagate the centromere identity, which assembles into a nucleosome together with histones H4, H2A and H2B without any known specific selection of DNA sequence [1, 2]. Centromeric chromatin is interspersed with CENP-A-nucleosomes and histone H3-containing nucleosomes, and it forms a repressive heterochromatin structure through the concerted action of various epigenetic mechanisms including histone methylation [2]. The plasticity of centromeric chromatin is crucial for the assembly of a large protein machine kinetochore that links chromosome to the spindle microtubule for mitotic segregation. Interestingly, recruitment of new CENP-A to centromere is not contemporaneous with replication of centromeric DNA in S phase. Rather, it is restricted to a brief interval in G1 immediately following mitosis in human cells [3] and slightly earlier in anaphase in the rapidly dividing Drosophila syncytial embryo. The assembly of new CENP-A nucleosomes in early G1 is dependent on CENP-C, Mis18 complex and HJURP (Holliday junction recognition protein). Among these proteins, HJURP is of great importance for CENP-A deposition as it is a CENPA-specific histone chaperone. HJURP interacts directly with CENP-A and histone H4 and recruits CENP-A to the centromere in a cell cycle-dependent manner. HJURP contains an approximately 80-amino acid CENP-A-binding domain (CBD) at the N terminus that is essential and sufficient for binding CENP-A. In addition, CENP-A contains a typical C-terminal histone fold domain, known as the centromere targeting domain (CATD), is required for interaction with HJURP [4]. In yeast, the HJURP homolog Scm3 is required for the deposition of centromeric nucleosomes. Down-regulation of HJURP impairs the deposition of newly synthesized CENP-A in mammalian cells. As a molecular chaperone of CENP-A, HJURP forms prenucleosomal complex with CENP-A/H4 heterodimer via its highly conserved Scm3 domain [4]. Recent studies show that mitotic master kinase Cdk1 phosphorylates HJURP at its C-terminal domain by which negatively regulates HJURP interaction with Mis18b and subsequent inhibits CENP-A loading in early mitosis when Cdk1 activity is high [5]. In fact, inhibition of Cdk1 resulted in premature C. Jin X. Yao (&) Anhui Key Laboratory for Cellular Dynamics & Chemical Biology, University of Science and Technology of China, Hefei 230027, China e-mail: yaoxb@ustc.edu.cn

Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores.

During mitosis, the spindle checkpoint senses kinetochores not properly attached to spindle microtubules and prevents precocious sister-chromatid separation and aneuploidy. The constitutive centromere-associated network (CCAN) at inner kinetochores anchors the KMN network consisting of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C) at outer kinetochores. KMN is a critical kinetochore receptor for both microtubules and checkpoint proteins. Here, we show that nearly complete inactivation of KMN in human cells through multiple strategies produced strong checkpoint defects even when all kinetochores lacked microtubule attachment. These KMN-inactivating strategies reveal multiple KMN assembly mechanisms at human mitotic kinetochores. In one mechanism, the centromeric kinase Aurora B phosphorylates Mis12C and strengthens its binding to the CCAN subunit CENP-C. In another, CENP-T contributes to KMN attachment in a CENP-H-I-K-dependent manner. Our study provides insights into the mechanisms of mitosis-specific assembly of the checkpoint platform KMN at human kinetochores.

Centromere Licensing: Mis18 Is Required to Polo-ver

The Mis18 complex is a critical player in determining when and where centromeres are built. A new study identifies Polo-like kinase (Plk1) as a positive regulator required for the localization of Mis18 to centromeres. This is a critical step that is essential for proper centromere function and maintaining the integrity of the genome.

Polo-like Kinase 1 Licenses CENP-A Deposition at Centromeres

To ensure the stable transmission of the genome during vertebrate cell division, the mitotic spindle must attach to a single locus on each chromosome, termed the centromere. The fundamental requirement for faithful centromere inheritance is the controlled deposition of the centromere-specifying histone, CENP-A. However, the regulatory mechanisms that ensure the precise control of CENP-A deposition have proven elusive. Here, we identify polo-like kinase 1 (Plk1) as a centromere-localized regulator required to initiate CENP-A deposition in human cells. We demonstrate that faithful CENP-A deposition requires integrated signals from Plk1 and cyclin-dependent kinase (CDK), with Plk1 promoting the localization of the key CENP-A deposition factor, the Mis18 complex, and CDK inhibiting Mis18complex assembly. By bypassing these regulated steps, we uncoupled CENP-A deposition from cell-cycle progression, resulting in mitotic defects. Thus, CENP-A deposition is controlled by a two-step regulatory paradigm comprised of Plk1 and CDK that is crucial for genomic integrity.

Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly

CENP-A chromatin forms the foundation for kinetochore assembly. Replication-independent incorporation of CENP-A at centromeres depends on its chaperone HJURPScm3, and Mis18 in vertebrates and fission yeast. The recruitment of Mis18 and HJURPScm3 to centromeres is cell cycle regulated. Vertebrate Mis18 associates with Mis18BP1KNL2, which is critical for the recruitment of Mis18 and HJURPScm3. We identify two novel fission yeast Mis18-interacting proteins (Eic1 and Eic2), components of the Mis18 complex. Eic1 is essential to maintain Cnp1CENP-A at centromeres and is crucial for kinetochore integrity; Eic2 is dispensable. Eic1 also associates with Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21, components of the constitutive CCAN/Mis6/Ctf19 complex. No Mis18BP1KNL2 orthologue has been identified in fission yeast, consequently it remains unknown how the key Cnp1CENP-A loading factor Mis18 is recruited. Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1KNL2, thus representing the functional counterpart of Mis18BP1KNL2 in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3HJURP Cnp1CENP-A loading factors. The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.

Mitotic Regulator Mis18β Interacts with and Specifies the Centromeric Assembly of Molecular Chaperone Holliday Junction Recognition Protein (HJURP)

The centromere is essential for precise and equal segregation of the parental genome into two daughter cells during mitosis. CENP-A is a unique histone H3 variant conserved in eukaryotic centromeres. The assembly of CENP-A to the centromere is mediated by Holliday junction recognition protein (HJURP) in early G1 phase. However, it remains elusive how HJURP governs CENP-A incorporation into the centromere. Here we show that human HJURP directly binds to Mis18β, a component of the Mis18 complex conserved in the eukaryotic kingdom. A minimal region of HJURP for Mis18β binding was mapped to residues 437-460. Depletion of Mis18β by RNA interference dramatically impaired HJURP recruitment to the centromere, indicating the importance of Mis18β in HJURP loading. Interestingly, phosphorylation of HJURP by CDK1 weakens its interaction with Mis18β, consistent with the notion that assembly of CENP-A to the centromere is achieved after mitosis. Taken together, these data define a novel molecular mechanism underlying the temporal regulation of CENP-A incorporation into the centromere by accurate Mis18β-HJURP interaction.

Modular Assembly of RWD Domains on the Mis12 Complex Underlies Outer Kinetochore Organization

Faithful chromosome segregation is mandatory for cell and organismal viability. Kinetochores, large protein assemblies embedded in centromeric chromatin, establish a mechanical link between chromosomes and spindle microtubules. The KMN network, a conserved 10-subunit kinetochore complex, harbors the microtubule-binding interface. RWD domains in the KMN subunits Spc24 and Spc25 mediate kinetochore targeting of the microtubule-binding subunits by interacting with the Mis12 complex, a KMN subcomplex that tethers directly onto the underlying chromatin layer. Here, we show that Knl1, a KMN subunit involved in mitotic checkpoint signaling, also contains RWD domains that bind the Mis12 complex and that mediate kinetochore targeting of Knl1. By reporting the first 3D electron microscopy structure of the KMN network, we provide a comprehensive framework to interpret how interactions of RWD-containing proteins with the Mis12 complex shape KMN network topology. Our observations unveil a regular pattern in the construction of the outer kinetochore.

Molecular characterization and clinical impact of t(11;15)(q23;q14-15) MLL-CASC5 rearrangement

Molecular characterization and clinical impact of t(11;15)(q23;q14-15) MLL-CASC5 rearrangement

βTrCP-mediated ubiquitylation regulates protein stability of Mis18β in a cell cycle-dependent manner

Ubiquitin E3 ligases including SCF complex are key regulators of cell cycle. Here, we show that Mis18β, a component of Mis18 complex governing CENP-A localization, is a new substrate of βTrCP-containing SCF complex. βTrCP interacted with Mis18β exclusively during interphase but not during mitosis and mediated proteasomal degradation of Mis18β leading to the inactivation of Mis18 complex during interphase. In addition, uncontrolled stabilization of Mis18β caused cell death. Together, we propose that βTrCP-mediated regulation of Mis18β stability is a mechanism to restrict centromere function of Mis18 complex from late mitosis to early G1 phase.

Stability of kinetochore‐microtubule attachment and the role of different KMN network components in Drosophila

Kinetochores bind spindle microtubules and also act as signaling centers that monitor this interaction. Defects in kinetochore assembly lead to chromosome missegregation and aneuploidy. The interaction between microtubules and chromosomes involves a conserved super-complex of proteins, known as the KNL1Mis12Ndc80 (KMN) network, composed by the KNL1 (Spc105), Mis12, and Ndc80 complexes. Previous studies indicate that all components of the network are required for kinetochore-microtubule attachment and all play relevant functions in chromosome congression, biorientation, and segregation. Here, we report a comparative study addressing the role of the different KMN components using dsRNA and in vivo fluorescence microscopy in Drosophila S2 cells allowing us to suggest that different KMN network components might perform different roles in chromosome segregation and the mitotic checkpoint signaling. Depletion of different components results in mostly lateral kinetochore-microtubule attachments that are relatively stable on depletion of Mis12 or Ndc80 but very unstable after Spc105 depletion. In vivo analysis on depletion of Mis12, Ndc80, and to some extent Spc105, shows that lateral kinetochore-microtubule interactions are still functional allowing poleward kinetochore movement. We also find that different KMN network components affect differently the localization of spindle assembly checkpoint (SAC) proteins at kinetochores. Depletion of Ndc80 and Spc105 abolishes the mitotic checkpoint, whereas depletion of Mis12 causes a delay in mitotic progression. Taken together, our results suggest that Mis12 and Ndc80 complexes help to properly orient microtubule attachment, whereas Spc105 plays a predominant role in the kinetochore-microtubule attachment as well as in the poleward movement of chromosomes, SAC response, and cell viability.

CENP-T provides a structural platform for outer kinetochore assembly

The kinetochore forms a dynamic interface with microtubules from the mitotic spindle during mitosis. The Ndc80 complex acts as the key microtubule-binding complex at kinetochores. However, it is unclear how the Ndc80 complex associates with the inner kinetochore proteins that assemble upon centromeric chromatin. Here, based on a high-resolution structural analysis, we demonstrate that the N-terminal region of vertebrate CENP-T interacts with the 'RWD' domain in the Spc24/25 portion of the Ndc80 complex. Phosphorylation of CENP-T strengthens a cryptic hydrophobic interaction between CENP-T and Spc25 resulting in a phospho-regulated interaction that occurs without direct recognition of the phosphorylated residue. The Ndc80 complex interacts with both CENP-T and the Mis12 complex, but we find that these interactions are mutually exclusive, supporting a model in which two distinct pathways target the Ndc80 complex to kinetochores. Our results provide a model for how the multiple protein complexes at kinetochores associate in a phospho-regulated manner.

Dynamic Nucleotide-dependent Interactions of Cysteine- and Histidine-rich Domain (CHORD)-containing Hsp90 Cochaperones Chp-1 and Melusin with Cochaperones PP5 and Sgt1

Mammals have two cysteine- and histidine-rich domain (CHORD)-containing Hsp90 cochaperones, Chp-1 and melusin, which are homologs of plant Rar1. It has been shown previously that Rar1 CHORD directly interacts with ADP bound to the nucleotide pocket of Hsp90. Here, we report that ADP and ATP can bind to Hsp90 cochaperones Chp-1 and PP5, inducing their conformational changes. Furthermore, we demonstrate that Chp-1 and melusin can interact with cochaperones PP5 and Sgt1 and with each other in an ATP-dependent manner. Based on the known structure of the Rar1-Hsp90 complex, His-186 has been identified as an important residue of Chp-1 for ADP/ATP binding. His-186 is necessary for the nucleotide-dependent interaction of Chp-1 not only with Hsp90 but also with Sgt1. In addition, Ca(2+), which is known to bind to melusin, enhances the interactions of melusin with Hsp90 and Sgt1. Furthermore, melusin acquires the ADP preference for Hsp90 binding in the presence of Ca(2+). Our newly discovered nucleotide-dependent interactions between cochaperones might provide additional complexity to the dynamics of the Hsp90 chaperone system, also suggesting potential Hsp90-independent roles for these cochaperones.

Plk1 Phosphorylates Sgt1 at the Kinetochores To Promote Timely Kinetochore-Microtubule Attachment

Accurate chromosome segregation during cell division maintains genomic integrity and requires the proper establishment of kinetochore-microtubule attachment in mitosis. As a key regulator of mitosis, Polo-like kinase 1 (Plk1) is essential for this attachment process, but the molecular mechanism remains elusive. Here we identify Sgt1, a cochaperone for Hsp90, as a novel Plk1 substrate during mitosis. We show that Sgt1 dynamically localizes at the kinetochores, which lack microtubule attachments during prometaphase. Plk1 is required for the kinetochore localization of Sgt1 and phosphorylates serine 331 of Sgt1 at the kinetochores. This phosphorylation event enhances the association of the Hsp90-Sgt1 chaperone with the MIS12 complex to stabilize this complex at the kinetochores and thus coordinates the recruitment of the NDC80 complex to form efficient microtubule-binding sites. Disruption of Sgt1 phosphorylation reduces the MIS12 and NDC80 complexes at the kinetochores, impairs stable microtubule attachment, and eventually results in chromosome misalignment to delay the anaphase onset. Our results demonstrate a mechanism for Plk1 in promoting kinetochore-microtubule attachment to ensure chromosome stability.

Putting CENP-A in its place

The centromere is the chromosomal region that directs kinetochore assembly during mitosis in order to facilitate the faithful segregation of sister chromatids. The location of the human centromere is epigenetically specified. The presence of nucleosomes that contain the histone H3 variant, CENP-A, are thought to be the epigenetic mark that indicates active centromeres. Maintenance of centromeric identity requires the deposition of new CENP-A nucleosomes with each cell cycle. During S-phase, existing CENP-A nucleosomes are divided among the daughter chromosomes, while new CENP-A nucleosomes are deposited during early G1. The specific assembly of CENP-A nucleosomes at centromeres requires the Mis18 complex, which recruits the CENP-A assembly factor, HJURP. We will review the unique features of centromeric chromatin as well as the mechanism of CENP-A nucleosome deposition. We will also highlight a few recent discoveries that begin to elucidate the factors that temporally and spatially control CENP-A deposition.

Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore

Kinetochores attach the replicated chromosomes to the mitotic spindle and orchestrate their transmission to the daughter cells. Kinetochore-spindle binding and chromosome segregation are mediated by the multi-copy KNL1(Spc105), MIS12(Mtw1) and NDC80(Ndc80)complexes that form the so-called KMN network. KMN-spindle attachment is regulated by the AuroraB(Ipl1) and MPS1(Mps1)kinases. It is unclear whether other mechanisms exist that support KMN activity during the cell cycle. Using budding yeast, we show that kinetochore protein Cnn1 localizes to the base of the Ndc80 complex and promotes a functionally competent configuration of the KMN network. Cnn1 regulates KMN activity in a spatiotemporal manner by inhibiting the interaction between its complexes. Cnn1 activity peaks in anaphase and is driven by the Cdc28, Mps1 and Ipl1 kinases.

Roles of Mis18α in Epigenetic Regulation of Centromeric Chromatin and CENP-A Loading

The Mis18 complex has been identified as a critical factor for the centromeric localization of a histone H3 variant, centromeric protein A (CENP-A), which is responsible for the specification of centromere identity in the chromosome. However, the functional role of Mis18 complex is largely unknown. Here, we generated Mis18α conditional knockout mice and found that Mis18α deficiency resulted in lethality at early embryonic stage with severe defects in chromosome segregation caused by mislocalization of CENP-A. Further, we demonstrate Mis18α's crucial role for epigenetic regulation of centromeric chromatin by reinforcing centromeric localization of DNMT3A/3B. Mis18α interacts with DNMT3A/3B, and this interaction is critical for maintaining DNA methylation and hence regulating epigenetic states of centromeric chromatin. Mis18α deficiency led to reduced DNA methylation, altered histone modifications, and uncontrolled noncoding transcripts in centromere region by decreased DNMT3A/3B enrichment. Together, our findings uncover the functional mechanism of Mis18α and its pivotal role in mammalian cell cycle.

Spatiotemporal dynamics of Spc105 regulates the assembly of the Drosophila kinetochore.

The formation of kinetochores shortly before each cell division is a prerequisite for proper chromosome segregation. The synchronous mitoses of Drosophila syncytial embryos have provided an ideal in vivo system to follow kinetochore assembly kinetics and so address the question of how kinetochore formation is regulated. We found that the nuclear exclusion of the Spc105/KNL1 protein during interphase prevents precocious assembly of the Mis12 complex. The nuclear import of Spc105 in early prophase and its immediate association with the Mis12 complex on centromeres are thus the first steps in kinetochore assembly. The cumulative kinetochore levels of Spc105 and Mis12 complex then determine the rate of Ndc80 complex recruitment commencing only after nuclear envelope breakdown. The carboxy-terminal part of Spc105 directs its nuclear import and is sufficient for the assembly of all core kinetochore components and CENP-C, when localized ectopically to centrosomes. Super-resolution microscopy shows that carboxy-terminus of Spc105 lies at the junction of the Mis12 and Ndc80 complexes on stretched kinetochores. Our study thus indicates that physical accessibility of kinetochore components plays a crucial role in the regulation of Drosophila kinetochore assembly and leads us to a model in which Spc105 is a licensing factor for its onset.

CENP-C facilitates the recruitment of M18BP1 to centromeric chromatin

Centromeres are important structural constituents of chromosomes that ensure proper chromosome segregation during mitosis by providing defined sites for kinetochore attachment. In higher eukaryotes, centromeres have no specific DNA sequence and thus, they are rather determined through epigenetic mechanisms. A fundamental process in centromere establishment is the incorporation of the histone variant CENP-A into centromeric chromatin, which provides a binding platform for the other centromeric proteins. The Mis18 complex, and, in particular, its member M18BP1 was shown to be essential for both incorporation and maintenance of CENP-A. Here we show that M18BP1 displays a cell cycle-regulated association with centromeric chromatin in mouse embryonic stem cells. M18BP1 is highly enriched at centromeric regions from late anaphase through to G1 phase. An interaction screen against 16 core centromeric proteins revealed a novel interaction of M18BP1 with CENP-C. We mapped the interaction domain in M18BP1 to a central region containing a conserved SANT domain and in CENP-C to the C-terminus. Knock-down of CENP-C leads to reduced M18BP1 association and lower CENP-A levels at centromeres, suggesting that CENP-C works as an important factor for centromeric M18BP1 recruitment and thus for maintaining centromeric CENP-A.